ES2634626T5 - Improved nisin production process - Google Patents

Description

DESCRIPTION

Improved nisin production process

Field of the invention

[0001] The present invention relates to fermentation processes. More particularly, an improved method is provided for the production of lactic acid starters, which also contain nisin.

Background of the invention

[0002] To prevent spoilage and deterioration of the quality of food products, preservatives are commonly added. For decades, chemically synthesized food preservatives have been the primary choice to achieve this goal. More recently, safety concerns regarding these chemically synthesized food preservatives have increased. Since then, there has been a growing interest in antimicrobial substances derived from traditional foods.

[0003] Lactic acid bacteria are useful microorganisms, which have traditionally been used in the production of various fermented food products. Lactic acid fermentation inhibits the growth of other contaminating bacteria. By reducing the pH of lactic acid production systems by lactic acid fermentation, spoilage and deterioration of the quality of such food products can be avoided and / or reduced. Additionally, it has been determined that some additional antimicrobial substances, such as bacteriocins, are produced by some lactic acid bacteria. A bacteriocin known to control pathogens in food is nisin. Nisin is produced by strains of Lactococcus lactis and inhibits a wide range of gram-positive bacteria. These bacteriocins belong to the group of lantibiotics (class I bacteriocins) and have a long history as food preservatives.

[0004] It is becoming more and more common to use ferments containing lactic acid and nisin produced by said strains as preservatives in all types of food products.

[0005] Conventional bacteriocin fermentation methods are generally inefficient when it comes to large-scale (commercial) nisin production due to limited production rates and unsatisfactory final nisin concentration. Prolonging the fermentation process inherently also increases the risk of contaminating production batches. Most importantly, slow nisin production rates make commercial nisin production expensive. Another relevant cost factor is the relatively expensive fermentation media required.

[0006] Nisin-containing ferments are only exceptionally produced in the same plant where they are applied to produce the final food products. In most cases, the nisin-containing ferments are manufactured in one plant to be normally used some time later in another plant in the production of the food product. Fermentation products, therefore, frequently need to be processed to create formulations that can be stored for a time without losing quality and / or activity, that can be transported efficiently and that are easy to handle and dose in the production of products. food. For economic reasons, it is often convenient to offer these products in a concentrated form. Such formulations can be suitable in liquid, semi-liquid or solid formulations, each type of formulation has its specific advantages and disadvantages. The most preferable choice will usually vary depending on whether it is one use or the other.

[0007] The possibilities of producing stable and easy-to-use liquid, semi-liquid and / or solid formulations of ferments containing nisin are largely determined by the thermodynamic behavior of the lactic acid salt (s), which constitute most of the ferment. This thermodynamic behavior establishes the conditions required for subsequent processing in the chosen formulation type and also determines which conditions may in turn have a significant impact on the nisin content / activity in the formulation.

Last but not least, the organoleptic, dietary and food regulatory requirements further establish limitations on fermentation, subsequent processing steps, and the types and amounts of substances to be used in fermentation.

[0009] J.S. VANT HUL ET AL: "Neutralization / recovery of lactic acid from Lactococcus lactis: effects on biomass, lactic acid, and nisin production", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 13, no.

5, 1997, pages 527-532, XP055071927, ISSN: 0959-3993, DOI: 10.1023 / A: 1018509207939 refers to a Study of the effects of lactic acid neutralization on the production of biomass, lactic acid and nisin during the fermentation of lactic acid bacteria (LAB) producing nisin. This indicates that pH regulation is necessary to achieve a high cell population, lactic acid production and nisin grade. Generally, pH control has been carried out by adding alkali to form acid salt. Through fed batch tests, the efficacy of NaOH, NH 4 OH and NH4 CO3 as neutralizing agents was compared. The fermentation medium used contained fine vinasse (comprising approx. 20% crude protein) and the fermenter was used at pH 6.5, 30 ° C. The cultures were incubated for 10 to 12 hours at 30 ° C before being used as a fermentor inoculum. In general, NH4 OH performed significantly better than NaOH or NH4 CO3 as a neutralizing agent. The fact that it supplied an additional source of nitrogen made it considered responsible for the increase in cell growth, the production of nisin and lactic acid observed in the vinasse-based medium.

[0010] DE VUYST L, VANDAMME EJ: "Nisin, A Lantibiotic Produced by Lactococcus Lactis Subsp. Lactis: Properties, Biosynthesis, Fermentation and Applications" In: DE VUYST L, VANDAMME EJ: "Bacteriocins of Lactic Acid Bacteria", 1994, Chapman Hall, XP008163106, pages 151-221 is the chapter of a manual dealing with the properties, biosynthesis, fermentation and applications of nisin. It reports that a fermentation with L. lactis of a medium based on glucose, yeast autolysate, potassium hydrogen phosphate, magnesium sulfate and sodium chloride resulted in nisin contents of 1000 IU / ml. The addition of four amino acids (as a nitrogen source) to a minimal medium significantly increased the production of nisin. Fermentation conditions normally include incubation at 28-30 ° C for 12-24 hours and pH control in the range of 5.0-6.8. Nisin was described as suitable for the preservation of food products.

[0011] PEREZ-GUERRA N ET AL; "Production of bacteriocins from Lactococcuslactis subsp. Lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 using mussel-processing wastes", BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, vol. 36, no. 2, 2002, pages 119-125, XP002699033 refers to a study of bacteriocin production of L. lactis CECT 539 using waste from mussel processing. The strains were grown in MRS broth, the working cultures were subcultured twice in liquid cultures in the same medium at 30 ° C before use. The hydrolyzed medium contained 0.65 g / l of nitrogen. An increase in nisin production was achieved in media buffered with 0.10 M potassium hydrogen phthalate / NaOH. The maximum growth of L. lactis was obtained after 15 hours of incubation. Nisin synthesis ceased when the pH of the medium decreased to less than 4.

[0012] KALRA M S, DUDANI A T: "Effect of calcium carbonate on nisin production in a milk culture", INDIAN JOURNAL OF DAIRY SCIENCE, vol. 261-15, 1973, pages 146-148, XP008163503, discloses the results of a study on the effect of calcium carbonate on the production of nisin in a milk culture. Three levels of CaCO3 were used, that is, 1.0, 2.0 and 3.0% and their addition allowed the neutralization of the lactic acid produced in the medium, which resulted in a small change in pH during the incubation, presented with values of approximately 5.5. In this way, nisin production can be increased.

In summary, manufacturers of preservative compositions containing nisin are faced with a number of interrelated technological challenges to optimize the process and satisfy the needs and requirements imposed by the specific (end) uses of the product, particularly with respect to the production rate. and performance, stabilization of activity, and processing into stable, easy-to-use formulations. It is understood that keeping manufacturing costs at an acceptable level is always a factor in process design.

The object of the invention is to provide methods for producing nisin-containing starters and to formulate such starters in preservatives. In particular, it is an object of the present invention to provide improved processes for producing nisin-containing ferments by simple and efficient (batch) culture that allows simple subsequent processing to be converted into either a liquid, semi-liquid or dry powder product, containing a content of nisin and sufficient stability, good handling properties and satisfactory organoleptic properties.

Summary of the invention

[0015] The present invention provides a process for the production of lactate ferment containing nisin and comprising the following steps:

a) providing a nutrient medium comprising a solution of a fermentable substrate and a nitrogen source in an aqueous medium;

b) inoculating said nutrient medium with lactic acid bacteria that produce nisin; and

c) incubate the inoculated nutrient medium under conditions favorable for the growth and / or metabolic activity of said lactic acid bacteria for a period sufficient to produce a fermentation broth containing a minimum of 20 g / l of lactate equivalents and / or less 1000 IU / ml of equivalent activity of nisin, during said period the pH of the fermentation broth is controlled by adding one or more alkalizing agents that comprise alkaline sodium and alkaline calcium salts, where the Na: Ca ratio (w / w) of the added salts it is in the range of 1/6 - 1/1, preferably in the range of 1/6 - 1/2.

[0016] In an advantageous embodiment of the invention, the nutrient medium comprises a milk base, preferably whey.

[0017] In an advantageous embodiment of the invention, the nutrient medium is inoculated with an inoculation medium comprising activated lactic acid bacteria.

[0018] In an advantageous embodiment of the invention, the process comprises the additional step d1) of acidifying the fermentation broth by adding an acid, preferably lactic acid, in an amount sufficient to reduce the pH of the fermentation broth. fermentation below 4.8.

In an advantageous embodiment of the invention, the process comprises the additional step d2) of inactivation of the lactic acid bacteria by subjecting the fermentation broth to a temperature of at least 40 ° C for a period of at least 10 minutes .

In an advantageous embodiment of the invention, the process comprises the additional step e) of concentrating the ferment obtained in step c) or d) until obtaining a dry substance content of at least 30% by weight, preferably in the range of 30-50% by weight, by evaporation.

In an advantageous embodiment of the invention, the process comprises the additional step f) of cooling the concentrate obtained in step e) to a temperature below 40 ° C.

[0022] In an advantageous embodiment of the invention, the process comprises the additional step e ') drying by atomization of the ferment obtained in step c) or d) to produce a loose powder.

[0023] The present invention also provides the ferments and fermented products obtainable by the method described above, where the process comprises the additional steps of:

d1) optionally adding an amount of acid to reduce the pH of the fermented broth to below 6;

d2) optionally heating the fermentation broth obtained after step c) or d1) at a temperature of at least 40 ° C for a period of at least 10 minutes;

e) subjecting the ferment obtained in step c), d1) or d2) to a drying step to produce a dry powder composition with a water content of less than 10% by weight;

where the ferment contains sodium and calcium ions in a Na: Ca ratio in the range 1/6 - 1/2, where the ferment contains at least 25% by weight of lactate, based on the weight of the dry solids , and where the amount of sodium and calcium cations exceeds 0.5 stoichiometric equivalent, where the stoichiometric equivalent refers to the combined amount of sodium and calcium cations that would theoretically be necessary to provide counter ions for all lactate anions , and its various applications as a preservative.

These and other aspects and embodiments of the invention will be apparent to those skilled in the art based on the following detailed description and experimental work described in the subsequent section.

Detailed description of the invention

[0025] As indicated here, the first stage a) of the process comprises the preparation of a nutrient medium. In this document, the term "nutrient medium" is used to refer to the medium in original form prepared for fermentation. The nutrient medium supplies the substrates and nutrients that lactic acid bacteria need to grow and produce the various fermentation products. The nutrient medium is normally an aqueous medium comprising a fermentable substrate, a nitrogen source and micronutrients, where lactic acid bacteria can grow and reproduce. In principle, any combination of a carbon source, a nitrogen source and micronutrients can be used as long as it promotes the growth of lactic acid bacteria.

The term 'fermentation broth' is used here to refer to the nutrient medium after inoculation with the lactic acid bacteria. Thus, in the strict sense, different types of "fermentation broths" can be distinguished based on the stage in which the fermentation has progressed: (i) nutrient media in the originally provided form that include bacteria directly after inoculation; (ii) nutrient media in the fermentation process where some or most of the originally provided nutrients have already been consumed and fermentation products including lactate that have been excreted in the media by bacteria; and, (iii) media that has been removed from the fermenter after part or all of the nutrients have been consumed.

[0027] As used herein, the term "fermentable substrate" refers to the source of carbon that is converted to another compound by the metabolic action of lactic acid bacteria. Mono-, di-, tri-, oligo- and polysaccharides can be used as the carbon source, in particular sugars such as glucose, sucrose, fructose, galactose and lactose and / or (hydrolysates) of starch. Preferably, in this invention, the substrate is the carbohydrate selected from the group consisting of lactose, sucrose, and glucose, most preferably lactose.

[0028] These carbohydrates can be derived from a variety of sources, such as dairy products derived from plants, fruits or vegetables, eg molasses, fruit or vegetable juices, fruit or vegetable pulp, etc. The invention can be practiced using one or more carbohydrates in partially or substantially purified form. Alternatively, the invention can be practiced using a raw material containing one or more carbohydrates.

The nutrient medium usually contains at least 1 g / l, more preferably 5-300 g / l, most preferably 10-80 g / l, of the carbohydrate.

[0030] Suitable examples of nitrogen sources include plant-derived protein, such as soy protein and pea protein, protein from dairy products, yeast or yeast extract, meat extract, various types of fermentation fungi, as well as hydrolysates of any of the proteins mentioned above. It is preferred that the nitrogen source comprises free amino acids and / or short peptides. Suitable nitrogen sources can be prepared by hydrolyzing a protein source. In one embodiment of the invention, the nitrogen source is selected from the group consisting of pea protein, yeast extracts and milk product protein hydrolysates, more preferably yeast extracts and milk product protein hydrolysates, especially hydrolyzate of casein.

The nutrient medium usually contains at least 0.1 g / l, more preferably 0.2-50 g / l, most preferably 0.5-20 g / l of the nitrogen source. These amounts refer to the total dry solids weight of the spiked material as the nitrogen source per liter of nutrient medium, as will be understood by those skilled in the art. Therefore, the nutrient medium normally contains at least 0.1 g / l, more preferably 0.2-50 g / l, most preferably 0.5-20 g / l of proteins, peptides and / or amino acids. free.

[0032] In some embodiment of the invention, the nutrient medium comprises other micronutrients that promote the metabolic growth action of lactic acid bacteria, such as vitamins, minerals, cofactors and / or other trace elements. Micronutrients are generally used in an index of at least 0.01% (w / v), preferably an index of between 0.1 and 2% (w / v) in the nutrient medium. Typically, carbohydrate sources and / or nitrogen sources that can be used in accordance with the invention inherently contain micronutrients.

[0033] A particularly preferred embodiment of the invention relates to the fermentation of a dairy product. As used herein, the term "dairy" refers to whole (animal) milk, components of milk as well as products derived from milk, such as whey, whey permeate, milk permeate, yogurt and quark and products derived from the preparation of yogurt and quark. In some preferred embodiments, by volume, the major component of the nutrient medium is the dairy product. In some embodiments, the nutrient medium comprises at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the volume of milk, for example, a selected product of whole milk, raw milk, skim milk, reconstituted milk, condensed milk, rehydrated milk and the like. In some embodiment, the nutrient medium comprises at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the dry solids weight of solids. of milk, for example, whole milk solids, raw milk solids, skim milk solids, whey and the like. In a particularly preferred embodiment, said milk solid is whey.

[0034] Another preferred embodiment of the invention relates to the fermentation of a product derived from fruit or vegetables, such as juices, pastes or pulp derived from fruit or vegetables. In some preferred embodiments, by volume, the major component of the nutrient medium is the fruit or vegetable derived product. Suitable examples include tomato, carrot, spinach, molasses, etc. In one embodiment Particularly preferred of the invention, the product derived from fruit or vegetables is a juice obtained by mechanical compression or by macerating the raw fruit or vegetable. In some embodiments, the fruit or vegetable derived product comprises at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the volume of the medium. nutritious. In some embodiments, the nutrient medium comprises at least 80%, at least 90%, at least 95%, at least 99% of the weight of the dry solids of fruit or vegetable solids.

[0035] As indicated here, the subsequent step b) comprises the inoculation of the nutrient medium with lactic acid bacteria that produce nisin.

According to this invention, "lactic acid bacteria" are capable of producing nisin. In some preferred embodiments, lactic acid bacteria belong to the order Lactobacillales, preferably to the genus Lactococcus. The nisin-producing lactic acid bacterium most preferably belongs to Lactococcus lactis ssp. lactis, and examples thereof include JCM 7638, ATCC 11454, NCDO 497, and IFO 12007. In preferred embodiments, lactic acid bacteria are lactic acid bacteria that have a GRAS status.

In some embodiments, the fermentation broth comprises a single species of nisin-producing lactic acid bacteria, while, in other embodiments, the fermentation broth comprises a mixture of strains of lactic acid bacteria, at least one of which is capable of producing nisin. In one embodiment, a mixture of nisin-producing lactic acid bacteria strains is used.

[0038] In one embodiment, the nutrient medium is inoculated with a fermentation broth comprising activated lactic acid bacteria. The term 'activated' is used in this case to indicate that the composition used to inoculate the nutrient medium comprises lactic acid bacteria in a metabolically active state. When bacteria are introduced into the nutrient medium during direct seeding, that is, in the form of a dry, liquid or frozen concentrate, the bacteria do not take effect immediately and take time to become active. This 'time lag', also called the 'latency period', may involve the reestablishment of the bacteria stored in the natural way (rehydration stage of the bacteria), the restoration of metabolic activity and / or adaptation to the new environment. In accordance with a preferred embodiment of this invention, the inoculation of the nutrient medium (production) is carried out with a fermentation broth comprising activated lactic acid bacteria, which typically shorten the latency period, which can significantly reduce the overall pot life time. fermentation. The use of activated lactic acid bacteria can also increase the reproduction rate during the exponential stage and / or the lactic acid index and / or nisin production during the exponential and / or fixed stages of the fermentation process.

[0039] In an embodiment of the invention, a fermentation broth comprising activated lactic acid bacteria is used for inoculation of the nutrient medium, where the broth comprises more than 12 g / l lactate equivalents, more preferably more than 14 g / l, most preferably more than 16 g / l. As used herein, the term "lactate equivalent" refers to the total of the free lactic acid and conjugate base (dissociated acid), as will be understood by those skilled in the art. The terms "lactic acid" and "free lactic acid" are used interchangeably herein to refer to the acid form. The term lactate refers to the dissociated form of lactic acid. The (dissolved) salt of lactate is also specifically referred to in this case as the "lactate salt".

[0040] In an embodiment of the invention, the fermentation broth comprising activated lactic acid bacteria used for the inoculation of the nutrient medium is prepared by incubating the lactic acid bacteria in an activation medium, which is similar in composition to the medium nutritious production. In one embodiment of the invention, the activation medium has the same composition as the production nutrient medium. In a particularly preferred embodiment of the invention, the activation medium is enriched compared to the production nutrient medium, preferably one or more nutrients applied in the production nutrient medium are used in excessive amounts in the activation medium, which which normally means that the concentration is at least 120% of the concentration used in the nutrient medium. More preferably, the activation medium comprises one or more of said nutrients in concentrations of at least 130% of the concentrations employed in the production nutrient medium, even more preferably at least 140%. In a particularly preferred embodiment of the invention, the nitrogen source is used in excessive amounts in the activation medium.

As will be understood by those skilled in the art, the fermentation broth comprising activated lactic acid bacteria can be prepared using standard equipment and methodology.

[0042] As indicated herein, the process comprises a step c), where the inoculated nutrient medium is incubated to produce a fermentation broth comprising lactate and nisin. The production of lactic acid ferments on a scale requires strict control of the fermentation conditions, in particular the pH. If the pH, the formation of lactic acid as a product of the metabolic action of lactic acid producing bacteria, is not controlled, the pH will decrease as fermentation proceeds. A decrease in pH below a critical value, depending on the microorganism used in the process, could negatively affect the metabolic process of the microorganism and finally stop the fermentation process. The exact critical value will depend on the specific set of incubation conditions. In general, it is preferable that, during the incubation steps, the pH of the fermentation broth is kept in the range of 4.5 to 8.0, preferably in the range of 4.8 to 6.5, more preferably in the range 5.0-6, eg 5.5, to avoid inhibition of lactate production. Inhibition of the lactate product is considered to have occurred when the amount of lactate produced in fermentation (per batch) does not increase more than about 3% with another incubation for a period of up to about twelve hours under the same conditions. This definition assumes that sufficient nutrients for lactate production are still available in the fermentation broth and applies to both batch and continuous operations.

[0043] As mentioned above, the prevention of the lowering of the pH below the critical value is carried out by adding one or more alkalizing agents comprising alkaline salts of sodium and calcium. Alkalinization, as used in this case, refers to the effect of reducing the amount of acid in a solution to any extent and therefore increasing the pH value. It does not imply an increase in pH above neutral pH. As such, the term 'alkalinization' is considered synonymous with and interchangeable with 'neutralization'. The term "alkaline salt" refers to an organic or inorganic salt of the respective alkali metal or alkaline earth metal, which reacts with an acid to accept hydrogen atoms and / or donate a pair of valence electrons, and, as such, can be Add to the fermentation broth to increase the pH to the desired value. Examples of the alkaline salts according to the invention include sodium and calcium hydroxides, sodium and calcium carbonates. In a preferred embodiment of the invention, one or more alkalizing agents comprise sodium hydroxide and calcium hydroxide.

[0044] In part, the invention resides in the finding that it is advantageous to use both sodium and calcium salts as alkalizing agent and especially to combine sodium and calcium salts in amounts resulting in a Na: Ca ratio (w / w ) in the range of 1/6 - 1/1. In a more preferred embodiment of the invention, a Na: Ca (w / w) ratio is in the range of 1/6 - 1/2, more preferably in the range of 1 / 5.5-1 / 2, 5, most preferably a Na: Ca (w / w) ratio in the range of 1 / 5-1 / 3, eg 1/4.

[0045] As those skilled in the art will understand, in the context of this invention it is not essential that the sodium and calcium salts are mixed or not prior to addition to the ferment. It is envisaged that the sodium and calcium salts are added separately to the ferment, which can be done simultaneously or subsequently. However, for practical reasons it is preferred to previously mix the sodium and calcium salts, as those skilled in the art will understand. Therefore, the present process normally comprises the addition of an alkalization comprising a mixture of alkaline salts of sodium and calcium in relative amounts that produce the aforementioned proportions of sodium and calcium ions. In an even more preferred embodiment of the invention, an alkalizing agent is added in the form of an aqueous solution of alkaline salts of sodium and calcium in relative amounts that produce the above-named proportions of sodium and calcium ions, for example, an aqueous solution of 2.5-20% (w / v) of sodium hydroxide, preferably 3-10% (w / v), for example, about 5% w / v, and 5-40% (w / v) calcium hydroxide, preferably 10-30% (w / v), for example about 20% (w / v).

[0046] It is also envisioned that other alkalizing agents may be used in conjunction with the alkaline salts of sodium and calcium. For example, other alkali or alkaline earth metal hydroxides, such as potassium hydroxide, can also be added. Normally, only other alkalizing agents are used in minor amounts. It is preferred that the alkali salts of sodium and calcium are used in such amounts that at least 75% of the total amount by mole of added alkali and alkaline earth metal ions is calcium and sodium, more preferably at least 80%, at the minus 85%, at least 90%, at least 95%, or at least 97%.

[0047] As those skilled in the art will understand, pH control typically involves the continuous or repeated administration of alkalizing agents. The process is preferably automated for precise control of the pH to a predetermined value.

Fermentation will generally be carried out at temperatures conventionally employed in lactic acid fermentation processes. Typically temperatures in the range of 20 ° C to 40 ° C will be used, preferably temperatures in the range of 20 ° C to 35 ° C, such as about 30 ° C.

[0049] Under the conditions described herein, fermentations on a production scale can be carried out with a duration normally varying between 10 and 24 hours, although the invention is not particularly limited in appearance. Shorter fermentation times are generally preferred as these inherently reduce the risks of contamination of the fermentation broth with other microbiological species.

[0050] Incubation can be performed using standard equipment for batch and / or continuous processing. Typically the process is carried out in a production fermenter (standard) comprising a vat with a volume of at least 250 L, at least 500 L or at least 1000 L.

In a preferred embodiment of the invention, the process is carried out in batches. In batch processing according to this invention, the lactate content of the fermentation broth will gradually increase as long as the lowering of the pH is avoided by the addition of one or more alkalizing agents. Even so, at some point, it is possible to "limit the lactate concentration", which is the lactate concentration (concentration of undissociated and dissociated lactic acid) at which, under a certain set of incubation conditions (pH, medium nutrient, temperature, degree of aeration), the subsequent production of lactate is inhibited. In other words, the growth and / or production of lactate material may be stopped due to the accumulation of one or more fermentation products, as well as in response to the decrease in pH resulting from the production of fermentation products, i.e. the fermentation reaction has a self-limiting point for the given set of incubation conditions. The present inventors determined that the nisin content normally peaks before the fermentation reaction has reached the point of self-limiting and may decrease if the fermentation continues above that point. In the present process, the incubation step is usually stopped before the point where the lactate equivalent concentration reaches 25 g / L, preferably 24 g / L, more preferably 23 g / L, more preferably 22 g / L. Preferably, the incubation step is allowed to proceed to the point where the lactate equivalent concentration exceeds 15 g / L, more preferably 17 g / L, more preferably 18 g / L, more preferably 19 g / L, as more preferably 20 g / l. The present process typically involves the production of a broth containing a final nisin concentration of at least 1000 IU / ml, preferably at least 2000 IU / ml, and more preferably at least 2500 IU / ml, for example about 3000 IU / ml.

[0052] In a preferred embodiment of this invention, the fermentation process is stopped by the addition of an amount of acid to the fermentation broth, in particular lactic acid, sufficient to stop lactate production. The process is therefore established as defined in the previous section, where step c) is followed by a step d1) that comprises adding an amount of acid to reduce the pH of the fermented broth to below 6, preferably by below 5.5, preferably below 5, for example 4.7. In a particularly preferred embodiment, said acid is lactic acid. Lactic acid is usually added in the form of a concentrated or dilute liquid, such as in the form of an aqueous solution with a lactic acid content of at least 100 g / l, although the invention is not particularly limited in this regard. This step of acidifying the fermentation broth by adding acid has been shown to generally significantly improve nisin production and / or improve nisin stability during subsequent treatments involving heating of the composition.

[0053] After incubation of the inoculated nutrient medium for a period sufficient to reach the desired lactate and nisin levels, optionally, after stopping the fermentation by adding an acid, the fermentation broth is normally subjected to another treatment whose purpose is the inactivation of lactic acid bacteria by heat treatment. A process is therefore provided as defined above, which comprises the additional step d2) of heating the fermentation broth obtained after step c) or d1) by subjecting it to a temperature of at least 40 ° C, at minus 45 ° C, or at least 50 ° C, for a period of at least 10, at least 20, at least 30, at least 40 or at least 50 minutes to inactivate lactic acid bacteria.

[0054] The fermentation broth obtained using the present invention can be processed into a suitable product formulation simply by removing the water, usually to obtain a paste. Lactate will be present primarily as a mixture of sodium and calcium salts and the present inventors have determined that these ferments can be ideally concentrated to dry solids contents of 30% by weight or more without the problem of product salts crystallizing. The present inventors further found that processing the ferment into a concentrated form according to this embodiment gave particularly advantageous results in terms of quality of the resulting product, especially in terms of (final) activity of nisin and / or organoleptic properties. Therefore, in an embodiment of the invention, a process is provided as described above, comprising the additional step e) of concentrating the ferment obtained in step c), d1) or d2) into a paste with a dry substance content of at least 30% by weight, preferably in the range of 30-50% by weight. Any conventional process can be used to carry out step e). In a preferred embodiment, step e) is carried out by subjecting the ferment obtained in step c), d1) or d2) to an environment with temperatures higher than that of the environment, normally at an environment of 55-70 ° C, and preferably at reduced pressure, for example at a pressure of 190-250 mbar. Conventional equipment, well known to those skilled in the art, can be used to carry out this step.

In one embodiment, following step e), the concentrated liquid or paste is rapidly cooled to a temperature below 40 ° C, preferably below 30 ° C, more preferably below 20 ° C, more preferably at a temperature below 10 ° C. The present inventors determined that this step significantly influences the quality of the resulting product, especially the (final) activity of nisin and / or the organoleptic properties, for example, compared to products that do not cool rapidly after step e) . Accordingly, an embodiment of the invention is provided that comprises a step f) where the concentrated liquid or the paste obtained in step e) is cooled to a temperature below 40 ° C, preferably below 30 ° C, plus preferably lower than 20 ° C, more preferably at a temperature lower than 10 ° C, usually by placing it in an environment with a temperature lower than room temperature for a period sufficient to carry out the aforementioned temperature decrease. In a particularly preferred embodiment, this step comprises placing the composition in a refrigeration or cooling equipment with a temperature lower than 15 ° C, lower than 10 ° C or lower than 5 ° C. Preferably, during this stage, the concentrated liquid or paste is agitated to avoid the accumulation of material in parts of the equipment.

[0056] Alternatively, the fermentation broth obtained using the present invention can be processed into a loose powder product by evaporation of substantially all of the water. Such powders, which contain anhydrous calcium lactate in combination with sodium lactate for the most part, combine high stability with excellent rehydration / dissolution behavior. In particular, the ferments obtained according to the invention, in this context, have superior properties compared to ferments containing only calcium lactate as the main lactate salt.

Therefore, in an embodiment of the invention, a process is provided as described above, comprising the additional step e ') of subjecting the ferment obtained in step c), d1) or d2) to a drying step to produce a dry powder composition with a water content of less than 10% by weight, preferably less than 5% by weight, more preferably less than 3.5% by weight. In one embodiment, step e ') comprises spray drying the ferment to produce a free-flowing powder product. Preferably, drying the aqueous liquid comprises spray drying the aqueous liquid. In the present method, before spray drying, the aqueous liquid can be concentrated by evaporation. Preferably, the aqueous liquid has a dry substance content of 1-80% by weight, most preferably 10-60% by weight when fed in a spray drying.

[0058] Alternatively, a powder can be produced by first drying the aqueous liquid to produce a dry residue, for example by rotary drum drying and subsequently reducing the size of the dry residue, for example by grinding, milling or cutting. In one embodiment of the invention, a ferment may be optionally concentrated and cooled according to steps e) and f) as described above, before being subjected to another drying step e ') to produce a dry powder composition. .

[0059] Due to the use of one or more alkalizing agents that comprise alkaline salts of sodium and calcium in their relative amounts mentioned above, the present invention allows the production of ferments, including ferments based on dairy products that do not need any process of clarification or other type of process aimed at eliminating certain components (solids) from the fermentation broth to obtain an acceptable preservative product suitable for food use. Therefore, in a preferred embodiment of the present invention, a process is provided as defined hereinabove, where the fermentation broth is not subjected to a treatment step where the dissolved or undissolved solid matter is eliminated from the fermented broth according to stage c), that is, independently of the application of optional stages d), e) and / or f).

Another aspect of the invention concerns a lactate ferment product obtainable through the processes as defined above, where the process comprises the additional steps of:

d1) optionally adding an amount of acid to reduce the pH of the fermented broth to below 6;

d2) optionally heating the fermentation broth obtained after step c) or d1) at a temperature of at least 40 ° C for a period of at least 10 minutes;

e) subjecting the ferment obtained in step c), d1) or d2) to a drying step to produce a dry powder composition with a water content of less than 10% by weight;

where the ferment contains sodium and calcium ions in a Na: Ca ratio in the range 1/6 - 1/2, where the ferment contains at least 25% by weight of lactate, based on the weight of the dry solids , Y where the amount of sodium and calcium cations exceeds 0.5 stoichiometric equivalent, where the stoichiometric equivalent refers to the combined amount of sodium and calcium cations that would theoretically be necessary to provide counter ions for all lactate anions. The use of one or more alkalizing agents described herein achieves a lactate-containing product mainly in the form of sodium and calcium lactate, which confers certain advantageous properties on the fermentation products, especially with respect to the ability to process such fermentation products. in various liquid, semi-liquid and solid formulations and / or with regard to the organoleptic properties of these fermentation products.

[0061] In a particularly preferred embodiment of the invention, the lactate yeast product contains at least 40% by weight of lactate, based on the weight of the dry solids, more preferably, at least 50% by weight . As will be understood, this includes all types of lactate, including free lactic acid as well as lactate containing salts.

[0062] Normally, in the lactate starter product of the invention, the main part of lactate is in the form of sodium and calcium salts. Therefore, in a preferred embodiment, a lactate ferment product is provided as defined herein, comprising lactate anions and sodium and calcium cations, where the amount of sodium and calcium cations exceeds 0.75 stoichiometric equivalent, preferably it exceeds 0.9 stoichiometric equivalent. Furthermore, in a preferred embodiment, a lactate ferment product is provided as defined herein comprising lactate and sodium anions and calcium cations, where the amount of sodium and calcium cations is equal to or less than 1.0 of the stoichiometric equivalent. As those skilled in the art will understand, the stoichiometric equivalent refers to the combined amount of sodium and calcium cations that would theoretically be necessary to provide counter ions for all lactate anions.

[0063] In a preferred embodiment of the invention, the ratio Na: Ca (w / w) is in the range of 1 / 5.5-1 / 2.5, most preferably a ratio Na: Ca (p / p) in the range of 1 / 5-1 / 3, for example, 1/4.

[0064] In a particularly preferred embodiment, as understood in the previous section, the lactate ferment product contains appreciable levels of nisin. Therefore, in a preferred embodiment of the invention, a lactate ferment product is provided as defined above containing at least 0.10 mg of nisin per g of dry substance, more preferably at least 0 20 mg of nisin per g of dry substance, most preferably at least 0.25 mg of nisin per g of dry substance. It should be understood that nisin actually refers to different variants of nisin, such as nisin A and nisin Z, that differ in their amino acid sequence, usually only by a single amino acid. The most common variant is nisin A. The type of nisin produced will depend solely on the choice of bacterial strain used for fermentation. Preferred strains in the context of the present invention will normally produce nisin A. Therefore, nisin is preferably nisin A. The named amounts of nisin, as will be understood by those skilled in the art, apply equally to all variants of nisin. Also, it should be noted that 1 microgram of nisin is assumed to equal 40 IU. Therefore, preferred levels of nisin correspond to activities of at least 400 IU, at least 800 IU and at least 1000 IU, all per g of dry substance, respectively.

[0065] Other aspects of the present invention concern the use of a lactate ferment product as defined in any of the previous sections for the preservation of a food product, especially a food product or a drink, more in particular a product selected from the group consisting of processed and natural cheeses, cooked meats, canned foods, seafood, alcoholic beverages, dairy desserts, dairy beverages / liquids, ready meals, dressings, sauces, flour products, liquid egg products , etc.; and to methods for preserving such food products that comprise the step of incorporating or applying to them a lactate ferment product as defined in any of the aforementioned sections. The lactate ferments of the invention are particularly useful for preservation against spoilage by a microorganism, especially a microorganism selected from the group consisting of Listeria spp, clostridium spp, lactic acid bacteria, and bacillus spp. It is within the scope of those skilled in the art to determine the most appropriate dosages for optimal protection against spoilage and to avoid negative impact on other quality aspects of the food product, especially organoleptic properties. As explained above, the present lactate ferment products can be applied in relatively high dosages without negative impact on organoleptic properties and salt content.

[0066] For a proper understanding of this document and its claims, it should be understood that the verb "understand" and its conjugations are used in their non-limiting sense to mean that the elements that follow the word are included, but the elements that not specifically mentioned are not excluded. Furthermore, the reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of these elements are present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an", thus, usually means "at least one".

The following examples are offered for illustrative use only, and are in no way intended to limit the scope of the present invention.

Examples

Example 1

[0068] Batch flasks (2 L) are used as reactors for fermentation. This reactor is vacuum sterilized for 20 min at 121 ° C. All cultures are performed aseptically. Whey powder at 5% w / w and yeast extract at 0.2% w / w (based on dry weight) is used as nutrient medium (pasteurized for 30 min at 80 ° C inside the reactor) in all fermentations. . A Ca (OH) ² / NaOH mixture (composition in the table below) is used to control the pH, which is kept at approximately 5.5.

[0069] A first batch flask containing the nutrient medium is inoculated with a glycerol raw material from Lactococcus lactis ATCC 11454 and incubated overnight at 30 ° C. The inoculum is 2.5% w / w. During the exponential growth stage, the ferment from the first batch flask is transferred as inoculum to the next 2 L reactor containing nutrient medium, which is under the same conditions. Fermentation in the second reactor is stopped after the lactate concentration has reached more than 25 g / l. About 10 ml of lactic acid is sufficient (80% dry solids) to reduce the pH to 4.7. The reactor is kept at 50 ° C for 1 hour for inactivation. After that, the ferment cools down. The final amount of nisin is around 3000 IU / ml.

[0070] The ferment can be further purified and / or concentrated according to methods known to a person skilled in the art. By evaporation, for example, a paste or concentrate can be prepared. By spray drying, for example, a powder can be prepared.

[0071] A paste produced by the evaporation of a ferment as the previous preparation, with a dry substance content of 35.2%, comprising nisin with an activity of 67064 IU / g (based on 100% MS).

[0072] Various properties of the ferment are examined, such as the stability of the activity of nisin in the ferment, as well as in its concentrates, and the organoleptic properties of the ferment. The ferment and concentrates have sufficient stability of nisin activity to allow the transport and storage of the product in accordance with usual industrial practice. The incorporation of the ferment and / or concentrates thereof in various food products in effective dosages produces organoleptic properties that are generally preferred in relation to those obtained with nisin ferments of the state of the art.

Example 2:

[0073] The lactic acid ferment is produced according to the procedure of Example 1. The raw ferment is not purified and processed into powder by spray drying.

The products with anhydrous calcium lactate were obtained by drying at a product temperature during the spray drying process of 90 ° C, while the air temperature varies between 165 and 180 ° C.

[0075] The products with calcium lactate pentahydrate were obtained using the spray granulation technique using a fluidized bed in continuous operation. The product temperature was 60 ° C, the fluidizing air temperature was 110 ° C.

[0076] For dissolution tests, 2 g of a powder were accurately weighed into a plastic cup and two glass beads with a diameter of 6 mm were added. The mixture was mixed by hand for 1 min. To avoid water absorption, the air was purged with dry nitrogen. Dissolution tests were performed by adding the powder sample to 198 g of demineralized water which was stirred with a magnetic stirrer. Such are the test conditions, that the sink conditions always hold (i.e. after complete dissolution of the material, the concentration in the liquid is less than about 30% of the saturation solubility). Times to reach 10%, 50%, 90% and 95% of complete dissolution were calculated.

[0077] For stability tests, samples are stored in hermetically sealed glass jars to prevent any moisture exchange. The samples have been stored at temperatures of 4 ° C, room temperature (around 18-22 ° C) and 40 ° C. The properties of the powders are visually evaluated at different times. The yeast powder is stable when it remains as a free-flowing powder.

[0078] It is possible to neutralize the fermentation broth using calcium hydroxide (Ca (OH ² ), sodium hydroxide (NaOH) or a combination thereof. This leads to a certain ratio of calcium to sodium (Ca / Na) in the product final. A solid product containing only Na is not suitable due to the high Na content and unacceptable hygroscopicity of the dry product. A solid product has been produced with anhydrous calcium lactate and a high Ca / Na ratio of 20. This The product had the disadvantage of having an unacceptable taste and a long dissolution time (95%) of 88 s.

[0079] A product with calcium lactate pentahydrate and a Ca / Na ratio of 3-4 was acceptable from the point of view of taste and Na content. The dissolution time (95%) of this product is around 30s, which is acceptable. However, the product lacks sufficient stability. This is because calcium lactate is present in the form of calcium lactate pentahydrate. Sodium lactate, which is also presumably present, was able to extract water of crystallization from calcium lactate pentahydrate crystals. This leads to the dissolution of the sodium lactate in the calcium lactate extracted water and, as a result, the formation of paste, even when packaged in aluminum sachets.

[0080] It has been found that a product with calcium lactate anhydrate and sodium lactate (in a Ca / Na ratio of 3) still has an acceptable dissolution time (95%) of 31 s. Also, it has a proven acceptable stability as no doughing occurred. The composition of this product was as follows:

Total nisin activity (based on 100% DM, IU / g) 59082

Lactic acid (% w / w) 51

Succinic acid (% w / w) 1.12

Total Nitrogen (% w / w) 1.6

Na (% w / w) 2.9

Ca (% w / w) 8.1

Lactose (% w / w) 11

pH (10%) 4.7

Example 3

[0081] To test the taste impact of calcium and sodium on neutralizing agents, model products have been prepared. Milk was used as the reference product. Different samples have been prepared consisting of milk to which nisin and / or lactate ferments have been added with different concentrations of calcium and sodium, as presented in the following table.

[0082] These samples were evaluated by a panel. The results are analyzed by principal component analysis and summarized in a Biplot (cf. B.S. Everitt, An R and S-Plus Companion to Multivariate Analysis). The Biplot is represented in figure 1.

[0083] The results clearly show that the use of sodium as the only counter ion in the neutralizing agent (based on NA) produces a salty taste, while the use of calcium as the only counter ion (based on Ca) produces a flavor astringent and bitter. Using both, both calcium and sodium, in a ratio of between approximately 1: 1 and 6: 1 w / w a much more neutral taste can be achieved.

Claims (15)

1. Process for the production of a lactate ferment containing nisin and comprising the consecutive stages of: a) providing a nutrient medium comprising a solution of a fermentable substrate and a nitrogen source in an aqueous medium; b) inoculating said nutrient medium with nisin-producing lactic acid bacteria; and c) incubating the inoculated nutrient medium under conditions favorable for the growth and / or metabolic activity of said lactic acid bacteria for a period sufficient to produce a fermentation broth containing at least 20 g / l of lactate equivalents and / or at least 1000 IU / ml of nisin equivalent activity, during said period the pH of the fermentation broth is controlled by adding one or more alkalizing agents that comprise alkaline salts of sodium and calcium, where the Na: Ca (w / w) ratio of the added salts is in the range of 1/6 - 1/1, preferably 1/6 - 1/2. 2. Process according to claim 1, wherein during step c) the pH of the fermentation broth is kept in the range of 4.8 to 6.0. 3. Process according to claim 1 or 2, wherein the nutrient medium comprises a dairy product. 4. Process according to any of the preceding claims, wherein step b) comprises the inoculation of said nutrient medium with an inoculation medium comprising activated lactic acid bacteria. Process according to any of the preceding claims, wherein the process comprises the additional step d1) of acidifying the fermentation broth by adding an acid, preferably lactic acid, in an amount sufficient to reduce the pH of the fermentation broth by up to below 4.8. Process according to any of the preceding claims, wherein the process comprises the additional step d2) of inactivating the lactic acid bacteria by subjecting the fermentation broth to a temperature of at least 40 ° C for a period of at least 10 minutes. 7. Process according to any of the preceding claims, wherein the process comprises the additional step e) of concentrating the ferment obtained in step c) or d) until obtaining a dry substance content of at least 30% by weight, preferably in the range 30-50% by weight, by evaporation. 8. Process according to any of the preceding claims, wherein the process comprises the additional step f) of cooling the concentrate obtained in step e) to a temperature below 40 ° C. Process according to any of the preceding claims, wherein the process comprises the additional step e ') of spray drying the ferment obtained in step c) or d) or to produce a loose powder. 10. Process according to claim 9, wherein the dry powder has a water content of less than 5% by weight. 11. Process according to any of the preceding claims, wherein the nutrient medium comprises whey. 12. Process according to any of the preceding claims, wherein the process does not comprise a step where undissolved material is removed from the fermented broth. 13. Ferment obtainable through the process defined in any of the preceding claims, where the process comprises the additional steps of: d1) optionally adding an amount of acid to reduce the pH of the fermented broth to below 6; d2) optionally heating the fermentation broth obtained after step c) or d1) at a temperature of at least 40 ° C for a period of at least 10 minutes; e) subjecting the ferment obtained in step c), d1) or d2) to a drying step to produce a dry powder composition with a water content of less than 10% by weight; where the ferment contains sodium and calcium ions in a Na: Ca ratio in the range 1/6 - 1/2, where the ferment contains at least 25% by weight of lactate, based on the weight of the dry solids , and where the amount of sodium and calcium cations exceeds 0.5 stoichiometric equivalent, where the stoichiometric equivalent refers to the combined amount of sodium and calcium cations that would theoretically be necessary to provide counter ions for all lactate anions . Ferment according to claim 13, wherein the ferment contains at least 0.25 mg of nisin per gram of dry substance. 15. Use of a ferment according to claim 13 or 14 for the preservation of a food product.